Connexin hemichannel inhibition ameliorates epidermal pathology in a mouse model of keratitis ichthyosis deafness syndrome

Mutations in five different genes encoding connexin channels cause eleven clinically defined human skin diseases. Keratitis ichthyosis deafness (KID) syndrome is caused by point mutations in the GJB2 gene encoding Connexin 26 (Cx26) which result in aberrant activation of connexin hemichannels. KID syndrome has no cure and is associated with bilateral hearing loss, blinding keratitis, palmoplantar keratoderma, ichthyosiform erythroderma and a high incidence of childhood mortality. Here, we have tested whether a topically applied hemichhanel inhibitor (flufenamic acid, FFA) could ameliorate the skin pathology associated with KID syndrome in a transgenic mouse model expressing the lethal Cx26-G45E mutation. We found that FFA blocked the hemichannel activity of Cx26-G45E in vitro, and substantially reduced epidermal pathology in vivo, compared to untreated, or vehicle treated control animals. FFA did not reduce the expression of mutant connexin hemichannel protein, and cessation of FFA treatment allowed disease progression to continue. These results suggested that aberrant hemichannel activity is a major driver of skin disease in KID syndrome, and that the inhibition of mutant hemichannel activity could provide an attractive target to develop novel therapeutic interventions to treat this incurable disease.


Results
FFA blocked mutant Cx26 hemichannels. We tested the ability of FFA to block hemichannel activity of the Cx26-G45E mutation in vitro. Control Xenopus oocytes injected with water showed negligible membrane current when depolarizing voltage steps were applied (Fig. 1a). As previously reported 6,8 , the Cx26-G45E mutation induced large membrane currents in single oocytes (Fig. 1b). Perfusion of 50 µM FFA blocked these hemichannel currents (Fig. 1c). Fitting of a dose response curve revealed that FFA blocked hemichannel currents induced by Cx26-G45E with an IC 50 ≈ 31 µM (Fig. 1d). Thus, FFA can inhibit aberrant hemichannel activity associated with Cx26 KID mutations in vitro.
Quantitative assessment of skin disease in transgenic mice. Mice with inducible expression of the human Cx26-G45E mutation in keratinocytes replicate the epidermal pathology of KID syndrome. They also express enhanced green fluorescent protein (EGFP) in keratinocytes 13,26 , allowing epidermal thickness to be evaluated using an In Vivo Imaging System (IVIS). Figure 2 shows the correlation between EGFP fluorescence and epidermal pathology following the induction of a Cx26-G45E transgenic mouse by doxycycline. Wild-type control and Cx26-G45E transgenic mice had normal skin prior to doxycycline induction (Fig. 2a,b). KID lesions appeared on the transgenic animal by day 2 of induction, and progressively worsened through day 8 (Fig. 2c,f). Whole body fluorescent images showed that prior to induction, the wild-type control and transgenic animals had similar low levels of background fluorescence (Fig. 2g,h). EGFP fluorescent intensity increased for the trans-  www.nature.com/scientificreports/ genic animal from days 2-8, and correlated spatially with the visible KID lesions (Fig. 2i-l). Histological sections (Fig. 2m,n) obtained after induction showed normal epidermis in the control mouse, whereas the Cx26-G45E transgenic animal displayed typical features of KID syndrome, including acanthosis, papillomatosis, and a thickened stratum corneum 13 . Frozen sections showed an absence of EGFP fluorescence in control skin (Fig. 2o), and a strong EGFP signal throughout the thickened epidermis of Cx26-G45E transgenic skin (Fig. 2p). Plotting the total fluorescent radiant efficiency against the time after doxycycline induction (Fig. 2q) demonstrated  www.nature.com/scientificreports/ an increase in fluorescence in the transgenic mouse that was temporally correlated with worsening pathology. Therefore, IVIS imaging of EGFP fluorescence provided a spatially and temporally correlated measure of epidermal pathology in Cx26-G45E transgenic mice. Variation in epidermal pathology was observed between individual Cx26-G45E mice, most likely due to their outbred and undefined genetic background 13,32 . Figure 3 shows representative examples of the variability in skin disease development. Wild-type control mice never exhibited erythrokeratoderma while on a doxycycline containing diet (Fig. 3a). All Cx26-G45E mice developed epidermal pathology, but the response varied from weak to strong (Fig. 3b-d). Similar trends were observed in the EGFP fluorescence data, which showed that all animals produced an increasing response higher than controls, but the slope and magnitude of the radiant efficiency varied (Fig. 3e). Despite inter-animal variability, the strong intra-animal correlation between the IVIS measurement of EGFP fluorescence and severity of disease was observed. To ensure that comparisons between untreated, drug, and vehicle treated mice were made between animals exhibiting similar pathology, we adopted the following experimental approach: Groups of transgenic mice were induced with doxycycline and subjected to daily IVIS imaging to select cohorts with similar levels of increase in total fluorescent radiant efficiency and disease progression. These cohorts were then divided equally among the different treatment groups in experiments testing the efficacy of FFA treatment.

Mobilisin treatment reduced epidermal pathology.
To test if topical treatment with FFA could ameliorate the skin disease associated with KID syndrome, we treated Cx26-G45E transgenic mice with topically applied FFA using commercially available Mobilisin cream (Fig. 4). Transgenic animals were induced with doxycycline containing chow and EGFP fluorescence was quantitatively monitored in the skin using IVIS imaging for 5 days to ensure equal disease progression between the control and Mobilisin treated groups. Animals were then taken off doxycycline and allowed to recover for 7 days, until KID lesions disappeared and EGFP fluorescence returned to baseline. Mice were then induced with doxycycline a second time, with or without twice daily treatment with Mobilisin beginning on the third day of the second induction. Visual observation showed a marked reduction of KID lesion severity in the Mobilisin treated animals compared to the untreated controls. Two weeks after the second doxycycline induction, the untreated Cx26-G45E animals (Fig. 4a) developed obvi- www.nature.com/scientificreports/ ous skin lesions that were greatly diminished in the animals receiving Mobilisin treatment (Fig. 4b), and these differences in lesions correlated well with the corresponding images of EGFP fluorescence (Fig. 4c,d). These findings were corroborated by the aggregate IVIS imaging data obtained for all eight mice (Fig. 4e,f). During the first induction on days 0 to 5, in the absence of hemichannel inhibition, the radiant efficiency increased in a similar manner for both groups of animals as disease progression worsened (P > 0.05, student's t-test). Between days 6 to 14, the fluorescence diminished to baseline following withdrawal of doxycycline. On days 19-28, the four animals topically treated with Mobilisin stabilized at a constant fluorescent level after treatment. In contrast, the radiant efficiency in the four untreated mice continued to increase as their skin lesions worsened, and the observed values were 47% higher than those in the Mobilisin treated group (P < 0.05). Skin biopsies were taken on day 28 and processed for histology. The untreated Cx26-G45E animals displayed typical KID pathology, that included extensive hyperkeratosis, osteal plugging, acanthosis, and papillomatosis compared to a negative control wild-type mouse (Fig. 4g,h). The Mobilisin treated animals ( Fig. 4i) still exhibited hyperkeratosis, but to a greatly reduced extent compared to the untreated animals. These data showed that a topically applied connexin hemichannel inhibitor significantly reduced epidermal thickening in a mouse model of KID syndrome.
FFA was required for reduced pathology. Emollients are used for palliative relief in KID syndrome 33 .
To confirm that non-specific effects of emollient were not responsible for the improved pathology seen in Fig. 4, we generated topical creams that were identical in composition with the exception of the presence or absence of FFA. FFA was dissolved in DMSO, mixed with Eucerin cream (final FFA concentration was 30 mM) and applied once daily. As controls, transgenic animals were either untreated, or treated with an equal volume of DMSO/ Eucerin vehicle (~ 0.5 ml/ mouse). Visual observation showed a reduction of KID lesion severity in the FFA  www.nature.com/scientificreports/ treated animal compared to either the untreated, or vehicle controls after 6 days of treatment (Fig. 5a-d). Skin biopsies obtained after 6 days of treatment confirmed that the vehicle control, or untreated Cx26-G45E animals displayed typical KID pathology. Similar to the results in Fig. 4, The FFA/Eucerin treated animal (Fig. 5f) had reduced skin pathology when compared to the untreated (Fig. 5e), or DMSO/Eucerin vehicle control (Fig. 5g) animals. These findings were supported by IVIS imaging data (Fig. 5h). Prior to topical treatment, the radiant efficiency increased in a similar manner for all three Cx26-G45E transgenic animals. Between days 5 to 10, the mouse topically treated with FFA/Eucerin stabilized at a constant fluorescent value. In contrast, the radiant efficiency in the untreated, or DMSO/Eucerin vehicle treated mice continued to increase as their skin lesions worsened. To confirm this result, we treated groups of animals (n = 4) with topical FFA/Eucerin and DMSO/Eucerin vehicle for 16 days (Fig. 5i,j). The mean radiant efficiency of EGFP fluorescence was reduced 50% in FFA/Eucerin treated animals (P < 0.05) after 16 days of topical application. These data showed that the improvement in skin pathology required the presence of the hemichannel inhibitor FFA.
FFA did not affect mutant connexin expression. Development of skin disease in the mouse model required induction of mutant connexin expression by doxycycline. This raised the concern that topical treatment with FFA may have interfered with the transgenic expression of mutant connexins, rather than pharmacologically inhibited their activity. To exclude this possibility, skin biopsies were obtained from wild-type control, untreated, and FFA/Eucerin treated transgenic animals and stained with an antibody against Cx26. Fluorescent microscopy showed no detectable EGFP or Cx26 signals in wild-type control epidermis (Fig. 6a). Both untreated (Fig. 6b) and FFA/Eucerin treated (Fig. 6c) Cx26-G45E transgenic mice showed a strong induction of EGFP in keratinocytes and a punctate pattern of Cx26 staining. These data showed that the improvement in skin pathology following FFA treatment was not due to off-target interference with the transgenic expression of the mutated connexin.

Cessation of FFA treatment allowed resumption of pathology. If inhibition of aberrant hemichan-
nel activity caused the arrest of skin disease development, then cessation of topical treatment with FFA should allow disease progression to continue. To qualitatively test this, we induced a Cx26-G45E transgenic animal, treated it for 6 days with topical FFA/Eucerin, and then suspended treatment and followed the mouse for 8 additional days. IVIS imaging (Fig. 6d) data revealed that prior to the FFA/Eucerin treatment, EGFP fluorescent efficiency steadily increased. During the FFA/Eucerin treatment, EGFP fluorescence remained at a stable level, and disease progression was arrested. When FFA/Eucerin treatment was terminated, the increase in fluorescent efficiency resumed. Comparison of images of the mouse during, and following cessation of treatment confirmed that the increase in EGFP fluorescence after FFA withdrawal correlated with increased epidermal pathology (Fig. 6e,f). These data suggested that continued topical treatment with FFA was required to sustain the amelioration of skin pathology associated with KID syndrome.

Discussion
KID syndrome is a rare disorder 34 . Infectious complications, increased cancer risk and respiratory dysfunction contribute to mortality among patients 22,24,35,36 . Current treatments for skin pathology include antibiotic/antifungal agents, retinoids, keratolytics, and surgical debridement 37,38 . We attempted to alleviate epidermal pathology using mechanistic insights derived from biophysical studies of the causative mutations 7,9,26 . We found that FFA inhibited mutant connexin hemichannels in vitro, and ameliorated the skin pathology associated with KID syndrome in a mouse model in vivo. It should be noted that obtaining direct evidence of hemichannel inhibition in the intact epidermis of a living mouse is extremely difficult and has not been done in this study. Nevertheless, these results suggested that hemichannel inhibition could provide a target to develop new therapeutic interventions 26,39,40 . They also supported the view that dysregulated hemichannel activity drives skin disease in KID syndrome. These ideas were reinforced by the recent observation that administration of a monoclonal antibody targeting connexin hemichannels to transgenic mice expressing a mutation causing Clouston syndrome reduced skin-related pathology 41 . The antibody blocked hemichannels in vitro, including those formed by mutated human connexins 42 . Antibody treatment curtailed epidermal hyperproliferation and alleviated hypertrophic sebaceous glands in a mouse model expressing the Cx30-A88V Clouston syndrome mutation, which displayed hemichananel activity 43,44 . This supported our results with FFA in a mouse model of KID syndrome, and could be applicable to other skin diseases. In addition to KID and Clouston syndromes, aberrant hemichannel activity has been linked to palmoplantar keratoderma and deafness, palmoplantar keratoderma and congenital alopecia-1, and erythrokeratodermia variabilis et progressive [45][46][47] .
Topical administration of FFA stopped progression of KID epidermal pathology, but did not eliminate it. While the reasons for this are unknown, it could be related to the impact of hemichannel activity on the epidermal barrier. In KID patients, an impaired barrier contributes to susceptibility to viral, bacterial, and fungal infection 36 , and could result from increased activity of connexin hemichannels 48,49 . The epidermis maintains a Ca 2+ gradient, and Ca 2+ is a regulator of keratinocyte differentiation 50 . KID hemichannels would disrupt this gradient, and potentially affect barrier formation [51][52][53] . This view is supported by mouse models of KID syndrome, where the epidermal calcium concentration was elevated in the cornified layer. The disturbed Ca 2+ gradient was associated with an altered lipid composition in the stratum corneum, which resulted in a defective barrier 54 . We speculate that as KID lesions develop in our inducible model, the barrier degrades which allows topically applied FFA to easily penetrate into the epidermis. As the FFA inhibits mutant Cx26 activity, hemichannel Ca 2+ flux is reduced, and the barrier recovers. Barrier recovery may reduce further FFA penetration into the epidermis, where inducible transgenic expression of mutant hemichannels would be ongoing. An equilibrium could result that  www.nature.com/scientificreports/ expression is constantly high, in contrast to human epidermis where Cx26 expression would be expected to decline as pathology improved 4,55,56 . Decades of research have led to the point where new treatments can now be based on mechanistic data. It has been established that connexin mutations augment hemichannel activity, and contribute to epidermal pathology through altered Ca 2+ flux 49,51,54,57 . Mouse models replicate human epidermal pathology 13,43,54,58 , and strategies to disrupt hemichannel activity have been developed 40,41,[59][60][61][62] . As hemichannel dysfunction may contribute to pathological mechanisms in other connexinopathies 21,40,60,61 , our finding that topical FFA treatment ameliorated the epidermal pathology of KID syndrome could have impact across additional hemichannel-dependent human disorders.
Hemichannel recording. Hemichannel currents were recorded 24 h after mRNA injection using a GeneClamp 500 amplifier, Digidata 1440A, and pClamp software (Axon, Foster City, CA). Electrodes (World Precision Instruments, Sarasota, FL) were pulled to 1-2 MΩ resistance (Narishige, Tokyo, Japan) and filled with 3 M KCl, 10 mM EGTA, and 10 mM HEPES, pH 7.4. Cells were recorded in MB medium without calcium 8 . Current-voltage curves were obtained by clamping cells at − 40 mV and imposing voltage steps in 10 mV increments from − 30 to + 60 mV 46 . Figure 6. FFA did not inhibit mutant connexin expression and cessation of treatment allowed progression of epidermal pathology. No EGFP or Cx26 signals were detected in wild-type control epidermis (a). Untreated (b) and FFA treated (c) transgenic mice showed an induction of EGFP in keratinocytes and a punctate pattern of Cx26 staining. A transgenic animal was treated for 6 days with FFA, and then treatment ended and the mouse was followed for an additional 8 days. IVIS imaging (d) showed that fluorescent efficiency increased prior to FFA treatment, remained stable during treatment, and resumed increasing after treatment was suspended. Images of the mouse taken during (e), and following cessation of FFA treatment (f) confirmed that FFA withdrawal correlated with increased epidermal pathology. www.nature.com/scientificreports/ Drug testing. Extracellular solutions were exchanged using gravity perfusion 26 . FFA (MilliporeSigma, Burlington, MA) was dissolved in DMSO at a concentration of 200 mM, and serially diluted into MB medium without calcium at concentrations between 1 and 500 µM. I-V curves were obtained before and after exchange of the extracellular medium with a known concentration of FFA. Percent inhibition was plotted against drug concentration and fit to a sigmoidal function (OriginLab, Northampton, MA) to determine the IC 50 .
Animals. Animal work was approved by the Stony Brook University IACUC, and conducted according to the NIH Guide for the Care and Use of Laboratory Animals and in compliance with the ARRIVE guidelines. Male and female Cx26-G45E transgenic mice in the SKH1 outbred and uncharacterized genetic background were used 13,32 , no sex based differences were observed 13 . When fed a doxycycline containing diet (625 mg/kg, Envigo, Indianapolis, IN), these mice express Cx26-G45E and enhanced green fluorescent protein (EGFP) as independent proteins in epidermal keratinocytes. Animals were genotyped by PCR amplification of tail genomic DNA (Choice Taq, Thompson Scientific, Swedesboro, NJ) as described 13 .
In-vivo fluorescent imaging. EGFP fluorescence was monitored in transgenic animals using an In Vivo Imaging System (Lumina III, Perkin Elmer, Melville, NY). Mice were anesthetized with isoflurane and whole body dorsal fluorescent images were acquired. EGFP fluorescence was recorded as the total radiant efficiency (p/ sec/cm 2 /sr/μW/cm 2 ) for each animal subject. Histology. Skin was fixed in 4% formaldehyde in PBS for 24 h at room temperature. Tissues were rinsed with PBS, dehydrated through an ethanol series, and embedded in paraffin. 2-3 µm sections were cut, deparaffinized, and stained with hematoxylin-eosin 13 .